Nitrogen transport metabolism

ABSTRACT

This invention relates to an isolated nucleic acid fragments encoding an ammonium transporter. The invention also relates to the construction of a recombinant DNA construct encoding all or a portion of ammonium transporters, in sense or antisense orientation, wherein expression of the recombinant DNA construct may alter levels of the ammonium transporter in a transformed host cell.

This application is a continuation-in-part of application Ser. No.10/033,109 filed on Dec. 28, 2001 which claims priority benefit of U.S.application Ser. No. 09/384,625 filed on Aug. 27, 1999 which claimspriority benefit of U.S. Provisional Application No. 60/098,248 filedAug. 28, 1998.

FIELD OF THE INVENTION

This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid fragments encodingammonium transporters in plants and seeds.

BACKGROUND OF THE INVENTION

Higher plants are autotrophic organisms that can synthesize all of theirmolecular components from inorganic nutrients obtained from the localenvironment. Nitrogen is a key element in many compounds present inplant cells. It is found in the nucleoside phosphates and amino acidsthat form the building blocks of nucleic acids and proteins,respectively. Availability of nitrogen for crop plants is an importantlimiting factor in agricultural production, and the importance ofnitrogen is demonstrated by the fact that only oxygen, carbon, andhydrogen are more abundant in higher plant cells. Nitrogen present inthe form of ammonia or nitrate is readily absorbed and assimilated byhigher plants.

Nitrate is the principal source of nitrogen that is available to higherplants under normal field conditions. Thus, the nitrate assimilationpathway is the major point of entry of inorganic nitrogen into organiccompounds (Hewitt et al. (1976) Plant Biochemistry, pp 633-6812, Bonner,and Varner, eds. Academic Press, NY). Although nitrate is generally themajor form of nitrogen available to plants, some plants directly utilizeammonia, under certain conditions.

In Saccharomyces cerevisiae, the transport of ammonium across the plasmamembrane for use as a nitrogen source is mediated by at least twofunctionally distinct transport systems. Expression of an ArabidopsiscDNA in a mutant yeast strain deficient in two ammonium uptake systemsallowed the identification of a plant ammonium transporter. The isolatedcDNA encodes a highly hydrophobic protein with 9-12 putative membranespanning regions. Sequence homologies to genes of bacterial and animalorigin indicated that this type of transporter is conserved over a broadrange of organisms suggesting that this gene encodes a high-affinityammonium transporter (Ninneman et al. (1994) EMBO J. 13:3464-3471). Agene encoding an ammonium transporter has been identified in yeast whichis most highly expressed when the cells are grown on low concentrationsof ammonium or on ‘poor’ nitrogen sources like urea or proline. Thisgene is down-regulated when the concentration of ammonium is high orwhen other ‘good’ nitrogen sources like glutamine or asparagine aresupplied in the culture medium. The main function of this gene appearsto be to enable cells grown under nitrogen-limiting conditions toincorporate ammonium present at relatively low concentrations in thegrowth medium (Marine et al. (1994) EMBO J. 13:3456-3463).

Genes encoding high affinity ammonium transporters have yet to beidentified in corn, soybean and wheat, although ESTs encoding peptideswith similarities to cDNAs encoding high-affinity ammonium transportersare found in the NCBI database. Rice ESTs having General Identifier Nos.568344 and 2309655 encode peptides with similarities to high-affinityammonium transporters. Genes encoding ammonium transporters have yet tobe identified in corn, rice, soybean and wheat, although an EST encodinga peptide with similarities to cDNAs encoding ammonium transporters isfound in the NCBI database having NCBI General Identifier No. 5005512.

SUMMARY OF THE INVENTION

The instant invention relates to isolated polynucleotides, eachpolynucleotide comprising a nucleotide sequence encoding a firstpolypeptide that has at least 90% identity based on Clustal method ofalignment compared to a polypeptide selected from the group consistingof a corn ammonium transporter polypeptide of SEQ ID NO:2, a soybeanammonium transporter polypeptide of SEQ ID NO:4, a wheat ammoniumtransporter polypeptide of SEQ ID NO:6, a corn ammonium transporter ofSEQ ID NO:8, a rice ammonium transporter SEQ ID NO:10, a soybeanammonium transporter of SEQ ID NO:12, and wheat ammonium transporter ofSEQ ID NO:14 and at least 95% identity based on Clustal method ofalignment compared to a full length corn ammonium transporter of SEQ IDNO:16. The present invention also comprises an isolated polynucleotidecomprising the complement of the nucleotide sequences described above.

The present invention relates to an isolated host cell comprising anisolated polynucleotide comprising a nucleotide sequence of at least 30contiguous nucleotides derived from a nucleotide sequence selected fromthe group consisting of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15 and thecomplement of such nucleotide sequences. Suitable host cells includeeucaryotic cells such as yeast, and plant cells and procaryotic cellssuch as bacteria. The present invention also relates to an isolated hostcell comprising an isolated polynucleotide comprising a nucleotidesequence encoding a first polypeptide that has at least 90% identitybased on Clustal method of alignment compared to a polypeptide selectedfrom the group consisting of a corn ammonium transporter polypeptide ofSEQ ID NO:2, a soybean ammonium transporter polypeptide of SEQ ID NO:4,a wheat ammonium transporter polypeptide of SEQ ID NO:6, a corn ammoniumtransporter of SEQ ID NO:8, a rice ammonium transporter SEQ ID NO:10, asoybean ammonium transporter of SEQ ID NO:12 and wheat ammoniumtransporter of SEQ ID NO:14 and at least 95% identity based on Clustalmethod of alignment compared to a full length corn ammonium transporterof SEQ ID NO:16. The present invention also comprises host cellscomprising an isolated polynucleotide comprising the complement of thenucleotide sequences described above.

The present invention relates to a recombinant DNA construct comprisingan isolated polynucleotide comprising a nucleotide sequence (and itscomplement) encoding a first polypeptide that has at least 90% identitybased on Clustal method of alignment compared to a polypeptide selectedfrom the group consisting of a corn ammonium transporter polypeptide ofSEQ ID NO:2, a soybean ammonium transporter polypeptide of SEQ ID NO:4,a wheat ammonium transporter polypeptide of SEQ ID NO:6, a corn ammoniumtransporter of SEQ ID NO:8, a rice ammonium transporter SEQ ID NO:10, asoybean ammonium transporter of SEQ ID NO:12 and wheat ammoniumtransporter of SEQ ID NO:14 and at least 95% identity based on Clustalmethod of alignment compared to a full length corn ammonium transporterof SEQ ID NO:16, operably linked to suitable regulation sequences. Thepresent invention also relates to a recombinant DNA construct comprisinga nucleotide sequence of at least 30 contiguous nucleotides derived froma nucleotide sequence selected from the group consisting of SEQ ID NO:1,3, 5, 7, 9, 11, 13, 15 and the complement of such nucleotide sequences.

The present invention relates to a virus, preferably a baculoviruscomprising an isolated polynucleotide comprising a nucleotide sequenceencoding a first polypeptide that has at least 90% identity based onClustal method of alignment compared to a polypeptide selected from thegroup consisting of a corn ammonium transporter polypeptide of SEQ IDNO:2, a soybean ammonium transporter polypeptide of SEQ ID NO:4, a wheatammonium transporter polypeptide of SEQ ID NO:6, a corn ammoniumtransporter of SEQ ID NO:8, a rice ammonium transporter SEQ ID NO:10, asoybean ammonium transporter of SEQ ID NO:12 and wheat ammoniumtransporter of SEQ ID NO:14 and at least 95% identity based on Clustalmethod of alignment compared to a full length corn ammonium transporterof SEQ ID NO:16. The present invention also relates to a virus comprisesan isolated polynucleotide comprising the complement of the nucleotidesequences described above. The present invention also relates to a viruscomprising a nucleotide sequence of at least 30 contiguous nucleotidesderived from a nucleotide sequence selected from the group consisting ofSEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15 and the complement of suchnucleotide sequences.

The present invention relates to an ammonium transporter polypeptidecomprising at least 90% homology based on Clustal method of alignmentcompared to a polypeptide selected from the group consisting of SEQ IDNO:2, 4, 6, 8, 10, 12 and 14 and at least 95% identity based on Clustalmethod of alignment compared to a full length corn ammonium transporterof SEQ ID NO:16.

The present invention relates to a process for producing an isolatedhost cell comprising a recombinant DNA construct of the presentinvention described above, the process comprising either transforming ortransfecting an isolated compatible host cell with a recombinant DNAconstruct of the present invention.

The present invention relates to a method of selecting an isolatedpolynucleotide that affects the level of expression of ammoniumtransporter polypeptide in a plant cell, the method comprising the stepsof:

constructing an isolated polynucleotide comprising a nucleotide sequenceof at least 30 contiguous nucleotides derived from a nucleotide sequenceselected from the group consisting of SEQ ID NO:1, 3, 5, 7, 9, 11, 13,15 and the complement of such nucleotide sequences;

introducing the isolated polynucleotide into a plant cell;

measuring the level of ammonium transporter polypeptide in the plantcell containing the polypeptide; and

comparing the level of ammonium transporter polypeptide in the plantcell containing the isolated polynucleotide with the level of ammoniumtransporter polypeptide in a plant cell that does not contain therecombinant DNA construct.

The present invention relate to a method of selecting an insolatedpolynucleotide that affects the level of expression of ammoniumtransporter polypeptide in a plant cell comprising the steps describedabove; however, the isolated polynucleotide comprises a nucleotidesequence encoding a first polypeptide that has at least 90% identitybased on Clustal method of alignment compared to a polypeptide selectedfrom the group consisting of a corn ammonium transporter polypeptide ofSEQ ID NO:2, a soybean ammonium transporter polypeptide of SEQ ID NO:4,a wheat ammonium transporter polypeptide of SEQ ID NO:6, a corn ammoniumtransporter of SEQ ID NO:8, a rice ammonium transporter SEQ ID NO:10, asoybean ammonium transporter of SEQ ID NO:12 and wheat ammoniumtransporter of SEQ ID NO:14 and at least 95% identity based on Clustalmethod of alignment compared to a full length corn ammonium transporterof SEQ ID NO:16. The above methods may also use isolated polynucleotidecomprising the complement of the nucleotide sequences described above.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

The invention can be more fully understood from the following detaileddescription and the accompanying Sequence Listing which form a part ofthis application.

Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. The sequence descriptions and SequenceListing attached hereto comply with the rules governing nucleotideand/or amino acid sequence disclosures in patent applications as setforth in 37 C.F.R. §1.821-1.825.

TABLE 1 Ammonium Transporters SEQ ID NO: Protein Clone Designation(Nucleotide) (Amino Acid) Corn high affinity cr1n.pk0169.g8 1 2 ammoniumtransporter Soybean high affinity sfl1.pk0070.e12 3 4 ammoniumtransporter Wheat high affinity wlm12.pk0020.b10 5 6 ammoniumtransporter Corn ammonium transporter p0126.cnlds55r 7 8 Rice ammoniumtransporter rl0n.pk083.f9 9 10 Soybean ammonium transportersrc3c.pk003.h14 11 12 Wheat ammonium transporter wlk8.pk0013.b6 13 14Corn ammonium transporter cnr1c.pk002.e24f:fis 15 16

The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Research 13:3021-3030 (1985) and in the BiochemicalJournal 219 (No. 2):345-373 (1984) which are herein incorporated byreference. The symbols and format used for nucleotide and amino acidsequence data comply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

In the context of this disclosure, a number of terms shall be utilized.As used herein, a “polynucleotide” is a nucleotide sequence such as anucleic acid fragment. A polynucleotide may be a polymer of RNA or DNAthat is single- or double-stranded, that optionally contains synthetic,non-natural or altered nucleotide bases. A polynucleotide in the form ofa polymer of DNA, may comprise one or more segments of cDNA, genomic DNAor synthetic DNA.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the polypeptide encoded by the nucleotide sequence. “Substantiallysimilar” also refers to nucleic acid fragments wherein changes in one ormore nucleotide bases does not affect the ability of the nucleic acidfragment to mediate alteration of gene expression by gene silencingthrough for example antisense or co-suppression technology.“Substantially similar” also refers to modifications of the nucleic acidfragments of the instant invention such as deletion or insertion of oneor more nucleotides that do not substantially affect the functionalproperties of the resulting transcript vis-à-vis the ability to mediategene silencing or alteration of the functional properties of theresulting protein molecule. It is therefore understood that theinvention encompasses more than the specific exemplary nucleotide oramino acid sequences and includes functional equivalents thereof.

For example, it is well known in the art that antisense suppression andco-suppression of gene expression may be accomplished using nucleic acidfragments representing less than the entire coding region of a gene, andby nucleic acid fragments that do not share 100% sequence identity withthe gene to be suppressed. Moreover, alterations in a nucleic acidfragment which result in the production of a chemically equivalent aminoacid at a given site, but do not affect the functional properties of theencoded polypeptide, are well known in the art. Thus, a codon for theamino acid alanine, a hydrophobic amino acid, may be substituted by acodon encoding another less hydrophobic residue, such as glycine, or amore hydrophobic residue, such as valine, leucine, or isoleucine.Similarly, changes which result in substitution of one negativelycharged residue for another, such as aspartic acid for glutamic acid, orone positively charged residue for another, such as lysine for arginine,can also be expected to produce a functionally equivalent product.Nucleotide changes which result in alteration of the N-terminal andC-terminal portions of the polypeptide molecule would also not beexpected to alter the activity of the polypeptide. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity of the encodedproducts. Consequently, polynucleotide comprising a nucleotide sequenceof at least 30 contiguous nucleotides derived from a nucleotide sequenceselected from the group consisting of SEQ ID NO:1, 3, 5, 7, 9, 11, 13and the complement of such nucleotide sequences may be used in methodsof selecting an isolated polynucleotide that affects the expression ofthe ammonium transporter polypeptide in a plant cell.

Moreover, substantially similar nucleic acid fragments may also becharacterized by their ability to hybridize. Estimates of such homologyare provided by either DNA-DNA or DNA-RNA hybridization under conditionsof stringency as is well understood by those skilled in the art (Hamesand Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford,U.K.). Stringency conditions can be adjusted to screen for moderatelysimilar fragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. Post-hybridizationwashes determine stringency conditions. One set of preferred conditionsuses a series of washes starting with 6×SSC, 0.5% SDS at roomtemperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30min. A more preferred set of stringent conditions uses highertemperatures in which the washes are identical to those above except forthe temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS wasincreased to 60° C. Another preferred set of highly stringent conditionsuses two final washes in 0.1×SSC, 0.1% SDS at 65° C.

Substantially similar nucleic acid fragments of the instant inventionmay also be characterized by the percent identity of the amino acidsequences that they encode to the amino acid sequences disclosed herein,as determined by algorithms commonly employed by those skilled in thisart. Preferred are those nucleic acid fragments whose nucleotidesequences encode amino acid sequences that are 80% identical to theamino acid sequences reported herein. More preferred nucleic acidfragments encode amino acid sequences that are 90% identical to theamino acid sequences reported herein. Most preferred are nucleic acidfragments that encode amino acid sequences that are 95% identical to theamino acid sequences reported herein. Sequence alignments and percentidentity calculations were performed using the Megalign program of theLASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).Multiple alignment of the sequences was performed using the Clustalmethod of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) withthe default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments using the Clustal method were KTUPLE1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-4100. In general, asequence of ten or more contiguous amino acids or thirty or morecontiguous nucleotides is necessary in order to putatively identify apolypeptide or nucleic acid sequence as homologous to a known protein orgene. Moreover, with respect to nucleotide sequences, gene-specificoligonucleotide probes comprising 30 or more contiguous nucleotides maybe used in sequence-dependent methods of gene identification (e.g.,Southern hybridization) and isolation (e.g., in situ hybridization ofbacterial colonies or bacteriophage plaques). In addition, shortoligonucleotides of 12 or more nucleotides may be used as amplificationprimers in PCR in order to obtain a particular nucleic acid fragmentcomprising the primers. Accordingly, a “substantial portion” of anucleotide sequence comprises a nucleotide sequence that will affordspecific identification and/or isolation of a nucleic acid fragmentcomprising the sequence. The instant specification teaches amino acidand nucleotide sequences encoding polypeptides that comprise one or moreparticular plant proteins. The skilled artisan, having the benefit ofthe sequences as reported herein, may now use all or a substantialportion of the disclosed sequences for purposes known to those skilledin this art. Accordingly, the instant invention comprises the completesequences as reported in the accompanying Sequence Listing, as well assubstantial portions of those sequences as defined above.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without affecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment comprising a nucleotide sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a nucleic acidfragment for improved expression in a host cell, it is desirable todesign the nucleic acid fragment such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Synthetic nucleic acid fragments” can be assembled from oligonucleotidebuilding blocks that are chemically synthesized using procedures knownto those skilled in the art. These building blocks are ligated andannealed to form larger nucleic acid fragments which may then beenzymatically assembled to construct the entire desired nucleic acidfragment. “Chemically synthesized”, as related to nucleic acid fragment,means that the component nucleotides were assembled in vitro. Manualchemical synthesis of nucleic acid fragments may be accomplished usingwell established procedures, or automated chemical synthesis can beperformed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of nucleotide sequence to reflect thecodon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Recombinant DNA construct” refers any gene thatis not a native gene, comprising regulatory and coding sequences thatare not found together in nature. Accordingly, a recombinant DNAconstruct may comprise regulatory sequences and coding sequences thatare derived from different sources, or regulatory sequences and codingsequences derived from the same source, but arranged in a mannerdifferent than that found in nature. “Endogenous gene” refers to anative gene in its natural location in the genome of an organism. A“foreign” gene refers to a gene not normally found in the host organism,but that is introduced into the host organism by gene transfer. Foreigngenes can comprise native genes inserted into a non-native organism, orrecombinant DNA constructs. A “transgene” is a gene that has beenintroduced into the genome by a transformation procedure.

“Coding sequence” refers to a nucleotide sequence that codes for aspecific amino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence which can stimulate promoter activity and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven comprise synthetic nucleotide segments. It is understood by thoseskilled in the art that different promoters may direct the expression ofa gene in different tissues or cell types, or at different stages ofdevelopment, or in response to different environmental conditions.Promoters which cause a nucleic acid fragment to be expressed in mostcell types at most times are commonly referred to as “constitutivepromoters”. New promoters of various types useful in plant cells areconstantly being discovered; numerous examples may be found in thecompilation by Okamuro and Goldberg (1989) Biochemistry of Plants15:1-82. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined,nucleic acid fragments of different lengths may have identical promoteractivity.

The “translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) MolecularBiotechnology 3:225).

The “3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptide by the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to an RNAtranscript that includes the mRNA and so can be translated into apolypeptide by the cell. “Antisense RNA” refers to an RNA transcriptthat is complementary to all or part of a target primary transcript ormRNA and that blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

The term “operably linked” refers to the association of two or morenucleic acid fragments on a single nucleic acid fragment so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein. “Overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. “Co-suppression”refers to the production of sense RNA transcripts capable of suppressingthe expression of identical or substantially similar foreign orendogenous genes (U.S. Pat. No. 5,231,020, incorporated herein byreference).

“Altered levels” refers to the production of gene product(s) intransgenic organisms in amounts or proportions that differ from that ofnormal or non-transformed organisms.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor” protein refers to theprimary product of translation of mRNA; i.e., with pre- and propeptidesstill present. Pre- and propeptides may be but are not limited tointracellular localization signals.

A “chloroplast transit peptide” is an amino acid sequence which istranslated in conjunction with a protein and directs the protein to thechloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear localization signal included (Raikhel (1992) PlantPhys. 100:1627-1632).

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference).

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook etal. Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”).

Nucleic acid fragments encoding at least a portion of several ammoniumtransporters have been isolated and identified by comparison of randomplant cDNA sequences to public databases containing nucleotide andprotein sequences using the BLAST algorithms well known to those skilledin the art. The nucleic acid fragments of the instant invention may beused to isolate cDNAs and genes encoding homologous proteins from thesame or other plant species. Isolation of homologous genes usingsequence-dependent protocols is well known in the art. Examples ofsequence-dependent protocols include, but are not limited to, methods ofnucleic acid hybridization, and methods of DNA and RNA amplification asexemplified by various uses of nucleic acid amplification technologies(e.g., polymerase chain reaction, ligase chain reaction).

For example, genes encoding other high affinity ammonium transporters orammonium transporters, either as cDNAs or genomic DNAs, could beisolated directly by using all or a portion of the instant nucleic acidfragments as DNA hybridization probes to screen libraries from anydesired plant employing methodology well known to those skilled in theart. Specific oligonucleotide probes based upon the instant nucleic acidsequences can be designed and synthesized by methods known in the art(Maniatis). Moreover, the entire sequences can be used directly tosynthesize DNA probes by methods known to the skilled artisan such asrandom primer DNA labeling, nick translation, or end-labelingtechniques, or RNA probes using available in vitro transcriptionsystems. In addition, specific primers can be designed and used toamplify a part or all of the instant sequences. The resultingamplification products can be labeled directly during amplificationreactions or labeled after amplification reactions, and used as probesto isolate full length cDNA or genomic fragments under conditions ofappropriate stringency.

In addition, two short segments of the instant nucleic acid fragmentsmay be used in polymerase chain reaction protocols to amplify longernucleic acid fragments encoding homologous genes from DNA or RNA. Thepolymerase chain reaction may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding plant genes. Alternatively,the second primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002)to generate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′ directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl.Acad. Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220).Products generated by the 3′ and 5′ RACE procedures can be combined togenerate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165).Consequently, a polynucleotide comprising a nucleotide sequence of atleast 30 contiguous nucleotides derived from a nucleotide sequenceselected from the group consisting of SEQ ID NO:1, 3, 5, 7, 9, 11, 13and the complement of such nucleotide sequences may be used in methodsof obtaining a nucleic acid fragment encoding a substantial portion ofan amino acid sequence encoding an ammonium transporter.

Availability of the instant nucleotide and deduced amino acid sequencesfacilitates immunological screening of cDNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can be then be used to screen cDNA expression libraries toisolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol.36:1; Maniatis).

The nucleic acid fragments of the instant invention may be used tocreate transgenic plants in which the disclosed polypeptides are presentat higher or lower levels than normal or in cell types or developmentalstages in which they are not normally found. This would have the effectof altering the level of nitrogen transport and accumulation in thosecells. Nitrogen deficiency in plants results in stunted growth, and manytimes in slender and often woody stems. In many plants the first signalof nitrogen deficiency is chlorosis (yellowing of the leaves).

Overexpression of the proteins of the instant invention may beaccomplished by first making a recombinant DNA construct in which thecoding region is operably linked to a promoter capable of directingexpression of a gene in the desired tissues at the desired stage ofdevelopment. For reasons of convenience, the recombinant DNA constructmay comprise promoter sequences and translation leader sequences derivedfrom the same genes. 3′ Non-coding sequences encoding transcriptiontermination signals may also be provided. The instant recombinant DNAconstruct may also comprise one or more introns in order to facilitategene expression.

Plasmid vectors comprising the instant recombinant DNA construct canthen be made. The choice of plasmid vector is dependent upon the methodthat will be used to transform host plants. The skilled artisan is wellaware of the genetic elements that must be present on the plasmid vectorin order to successfully transform, select and propagate host cellscontaining the recombinant DNA construct. The skilled artisan will alsorecognize that different independent transformation events will resultin different levels and patterns of expression (Jones et al. (1985) EMBOJ. 4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78-86),and thus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, Western analysis of protein expression, or phenotypicanalysis.

For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the recombinant DNAconstruct described above may be further supplemented by altering thecoding sequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. PlantPhys. Plant Mol. Biol. 42:21-53), or nuclear localization signals(Raikhel (1992) Plant Phys. 100:1627-1632) added and/or with targetingsequences that are already present removed. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of utility may be discovered in the future.

It may also be desirable to reduce or eliminate expression of genesencoding the instant polypeptides in plants for some applications. Inorder to accomplish this, a recombinant DNA construct designed forco-suppression of the instant polypeptide can be constructed by linkinga gene or gene fragment encoding that polypeptide to plant promotersequences. Alternatively, a recombinant DNA construct designed toexpress antisense RNA for all or part of the instant nucleic acidfragment can be constructed by linking the gene or gene fragment inreverse orientation to plant promoter sequences. Either theco-suppression or antisense recombinant DNA constructs could beintroduced into plants via transformation wherein expression of thecorresponding endogenous genes are reduced or eliminated.

Molecular genetic solutions to the generation of plants with alteredgene expression have a decided advantage over more traditional plantbreeding approaches. Changes in plant phenotypes can be produced byspecifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression ofspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

The person skilled in the art will know that special considerations areassociated with the use of antisense or cosuppression technologies inorder to reduce expression of particular genes. For example, the properlevel of expression of sense or antisense genes may require the use ofdifferent recombinant DNA constructs utilizing different regulatoryelements known to the skilled artisan. Once transgenic plants areobtained by one of the methods described above, it will be necessary toscreen individual transgenics for those that most effectively displaythe desired phenotype. Accordingly, the skilled artisan will developmethods for screening large numbers of transformants. The nature ofthese screens will generally be chosen on practical grounds, and is notan inherent part of the invention. For example, one can screen bylooking for changes in gene expression by using antibodies specific forthe protein encoded by the gene being suppressed, or one could establishassays that specifically measure enzyme activity. A preferred methodwill be one which allows large numbers of samples to be processedrapidly, since it will be expected that a large number of transformantswill be negative for the desired phenotype.

The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to the these proteins by methodswell known to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptides are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct a recombinant DNA construct for production of the instantpolypeptides. This recombinant DNA construct could then be introducedinto appropriate microorganisms via transformation to provide high levelexpression of the encoded ammonium transporter. An example of a vectorfor high level expression of the instant polypeptides in a bacterialhost is provided (Example 7).

Additionally, the instant polypeptides can be used as targets tofacilitate design and/or identification of inhibitors of those enzymesthat may be useful as herbicides. This is desirable because thepolypeptides described herein catalyze various steps in nitrogen uptake.Accordingly, inhibition of the activity of one or more of the enzymesdescribed herein could lead to inhibition of plant growth. Thus, theinstant polypeptides could be appropriate for new herbicide discoveryand design.

All or a substantial portion of the nucleic acid fragments of theinstant invention may also be used as probes for genetically andphysically mapping the genes that they are a part of, and as markers fortraits linked to those genes. Such information may be useful in plantbreeding in order to develop lines with desired phenotypes. For example,the instant nucleic acid fragments may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Maniatis) ofrestriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet.32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4(1):37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences mayalso be used for physical mapping (i.e., placement of sequences onphysical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: APractical Guide, Academic press 1996, pp. 319-346, and references citedtherein).

In another embodiment, nucleic acid probes derived from the instantnucleic acid sequences may be used in direct fluorescence in situhybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154).Although current methods of FISH mapping favor use of large clones(several to several hundred KB; see Laan et al. (1995) Genome Research5:13-20), improvements in sensitivity may allow performance of FISHmapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic andphysical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997)Nature Genetics 7:22-28) and Happy Mapping (Dear and Cook (1989) NucleicAcid Res. 17:6795-6807). For these methods, the sequence of a nucleicacid fragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

Loss of function mutant phenotypes may be identified for the instantcDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci. USA 86:9402-9406; Koes et al.(1995) Proc. Natl. Acad. Sci. USA 92:8149-8153; Bensen et al. (1995)Plant Cell 7:75-84). The latter approach may be accomplished in twoways. First, short segments of the instant nucleic acid fragments may beused in polymerase chain reaction protocols in conjunction with amutation tag sequence primer on DNAs prepared from a population ofplants in which Mutator transposons or some other mutation-causing DNAelement has been introduced (see Bensen, supra). The amplification of aspecific DNA fragment with these primers indicates the insertion of themutation tag element in or near the plant gene encoding the instantpolypeptides. Alternatively, the instant nucleic acid fragment may beused as a hybridization probe against PCR amplification productsgenerated from the mutation population using the mutation tag sequenceprimer in conjunction with an arbitrary genomic site primer, such asthat for a restriction enzyme site-anchored synthetic adaptor. Witheither method, a plant containing a mutation in the endogenous geneencoding the instant polypeptides can be identified and obtained. Thismutant plant can then be used to determine or confirm the naturalfunction of the instant polypeptides disclosed herein.

EXAMPLES

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions.

Example 1 Composition of cDNA Libraries; Isolation and Sequencing ofcDNA Clones

cDNA libraries representing mRNAs from various corn, rice, soybean andwheat tissues were prepared. The characteristics of the libraries aredescribed below.

TABLE 2 cDNA Libraries from Corn, Rice, Soybean and Wheat Library TissueClone cr1n Corn Root From 7 Day Old Seedlings* cr1n.pk0169.g8 cnr1cPlants were Nitrogen starved until all seed reserves werecnr1c.pk002.e24.f:fis depleted of a Nitrogen source. Plants were inducedwith addition of Nitrogen, then samples were collected at 30 min- 1 hrand 2 hr after Nitrogen. p0126 Corn Leaf Tissue From V8-V10 Stages**,Night-Harvested, p0126.cnlds55r Pooled rl0n Rice 15 Day Old Leaf*r10n.pk083.f9 sfl1 Soybean Immature Flower sfl1.pk0070.e12 src3c Soybean8 Day Old Root Infected With Cyst Nematode src3c.pk003.h14 wlk8 WheatSeedlings 8 Hours After Treatment With Herbicide*** wlk8.pk0013.b6 Wlm12Wheat Seedlings 12 Hours After Inoculation With Erysiphewlm12.pk0020.b10 graminis f. sp tritici *These libraries were normalizedessentially as described in U.S. Pat. No. 5,482,845, incorporated hereinby reference. **Corn developmental stages are explained in thepublication “How a corn plant develops” from the Iowa State UniversityCoop. Ext. Service Special Report No. 48 reprinted June 1993.***Application of 6-iodo-2-propoxy-3-propyl-4(3H)-quinazolinone;synthesis and methods of using this compound are described in USSN08/545,827, incorporated herein by reference.

cDNA libraries may be prepared by any one of many methods available. Forexample, the cDNAs may be introduced into plasmid vectors by firstpreparing the cDNA libraries in Uni-ZAP™ XR vectors according to themanufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.).The Uni-ZAP™ XR libraries are converted into plasmid libraries accordingto the protocol provided by Stratagene. Upon conversion, cDNA insertswill be contained in the plasmid vector pBluescript. In addition, thecDNAs may be introduced directly into precut Bluescript II SK(+) vectors(Stratagene) using T4 DNA ligase (New England Biolabs), followed bytransfection into DH10B cells according to the manufacturer's protocol(GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors,plasmid DNAs are prepared from randomly picked bacterial coloniescontaining recombinant pBluescript plasmids, or the insert cDNAsequences are amplified via polymerase chain reaction using primersspecific for vector sequences flanking the inserted cDNA sequences.Amplified insert DNAs or plasmid DNAs are sequenced in dye-primersequencing reactions to generate partial cDNA sequences (expressedsequence tags or “ESTs”; see Adams et al., (1991) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

Example 2 Identification of cDNA Clones

cDNA clones encoding ammonium transporters were identified by conductingBLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol.Biol. 215:403-410;) searches for similarity to sequences contained inthe BLAST “nr” database (comprising all non-redundant GenBank CDStranslations, sequences derived from the 3-dimensional structureBrookhaven Protein Data Bank, the last major release of the SWISS-PROTprotein sequence database, EMBL, and DDBJ databases). The cDNA sequencesobtained in Example 1 were analyzed for similarity to all publiclyavailable DNA sequences contained in the “nr” database using the BLASTNalgorithm provided by the National Center for Biotechnology Information(NCBI). The DNA sequences were translated in all reading frames andcompared for similarity to all publicly available protein sequencescontained in the “nr” database using the BLASTX algorithm (Gish andStates (1993) Nature Genetics 3:266-272) provided by the NCBI. Forconvenience, the P-value (probability) of observing a match of a cDNAsequence to a sequence contained in the searched databases merely bychance as calculated by BLAST are reported herein as “pLog” values,which represent the negative of the logarithm of the reported P-value.Accordingly, the greater the pLog value, the greater the likelihood thatthe cDNA sequence and the BLAST “hit” represent homologous proteins.

Example 3 Characterization of cDNA Clones Encoding High AffinityAmmonium Transporter

The BLASTX search using the EST sequences from clones listed in Table 3revealed similarity of the polypeptides encoded by the cDNAs to highaffinity ammonium transporter from Oryza sativa and Arabidopsis thaliana(NCBI General Identifier Nos. 2160782 and 1703292, respectively). Shownin Table 3 are the BLAST results for the sequences of the entire cDNAinserts comprising the indicated cDNA clones (“FIS”):

TABLE 3 BLAST Results for Sequences Encoding Polypeptides Homologous toHigh Affinity Ammonium Transporter NCBI General BLAST pLog Clone StatusIdentifier No. Score cr1n.pk0169.g8 FIS 2160782 72.50cnr1c.pk002.e24.f:fis CGS 32488298 215.40 sfl1.pk0070.e12 FIS 1703292254.00 wlm12.pk0020.b10 FIS 2160782 254.00

Nucleotides 6 through 272 from clone w1 m12.pk0020.b10 are 91% identicalto nucleotides 64 through 330 of a 334 nucleotide rice EST having NCBIGeneral Identifier No. 568344. Nucleotides 6 through 98 from clone w1m12.pk0020.b10 are 90% identical to nucleotides 71 through 163 of a 305nucleotide rice EST having NCBI General Identifier No. 2309655.Nucleotides 116 through 236 from clone w1 m12.pk0020.b10 are 93%identical to nucleotides 181 through 301 of a 305 nucleotide rice ESThaving NCBI General Identifier No. 2309655. Nucleotides 1 through 1753from clone cnr1c.pk002.e24.f:fis are 83% identical to nucleotides 5through 1768 of a 2044 nucleotide rice sequence having NCBI GeneralIdentifier No. 32983741.

The data in Table 4 represents a calculation of the percent identity ofthe amino acid sequences set forth in SEQ ID NOs:2, 4, 6 and 16 and theOryza sativa sequences (NCBI General Identifier Nos. 2160782 and32488298 and the Arabidopsis thaliana sequence (NCBI General IdentifierNo 1703292)

TABLE 4 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toHigh Affinity Ammonium Transporter Percent Identity to SEQ ID NO.2160782 1703292 32488298 2 70.5 59.6 84.8 4 66.2 77.8 72.5 6 83.6 72.791.3 16 84.0 72.5 91.8

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode a portion of a corn, an entire soybean and an entire wheat highaffinity ammonium transporter. These sequences represent the first corn,soybean and wheat sequences encoding high affinity ammonium transporter.

Example 4 Characterization of cDNA Clones Encoding Ammonium Transporter

The BLASTX search using the EST sequences from clones listed in Table 5revealed similarity of the polypeptides encoded by the cDNAs to ammoniumtransporter from Arabidopsis thaliana (NCBI General Identifier No.3335376). Shown in Table 5 are the BLAST results for individual ESTs(“EST”), or the sequences of the entire cDNA inserts comprising theindicated cDNA clones (“FIS”):

TABLE 5 BLAST Results for Sequences Encoding Polypeptides Homologous toAmmonium Transporter BLAST pLog Clone Status Score 3335376p0126.cnlds55r EST 28.30 rl0n.pk083.f9 FIS 254.00 src3c.pk003.h14 FIS254.00 wlk8.pk0013.b6 FIS 254.00

The data in Table 6 represents a calculation of the percent identity ofthe amino acid sequences set forth in SEQ ID NOs:8, 10, 12 and 14 andthe Arabidopsis thaliana sequence (NCBI General Identifier No. 3335376).

TABLE 6 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toAmmonium Transporter Percent Identity to SEQ ID NO. 3335376 8 76.2 1068.2 12 75.6 14 64.0

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode a portion of a corn, an entire rice, an entire soybean, and anentire wheat ammonium transporter. These sequences represent the firstcorn, rice, soybean and wheat sequences encoding ammonium transporters.

Example 5 Expression of recombinant DNA Constructs in Monocot Cells

A recombinant DNA construct comprising a cDNA encoding the instantpolypeptides in sense orientation with respect to the maize 27 kD zeinpromoter that is located 5′ to the cDNA fragment, and the 10 kD zein 3′end that is located 3′ to the cDNA fragment, can be constructed. ThecDNA fragment of this gene may be generated by polymerase chain reaction(PCR) of the cDNA clone using appropriate oligonucleotide primers.Cloning sites (NcoI or SmaI) can be incorporated into theoligonucleotides to provide proper orientation of the DNA fragment wheninserted into the digested vector pML103 as described below.Amplification is then performed in a standard PCR. The amplified DNA isthen digested with restriction enzymes NcoI and SmaI and fractionated onan agarose gel. The appropriate band can be isolated from the gel andcombined with a 4.9 kb NcoI-SmaI fragment of the plasmid pML103. PlasmidpML103 has been deposited under the terms of the Budapest Treaty at ATCC(American Type Culture Collection, 10801 University Blvd., Manassas, Va.20110-2209), and bears accession number ATCC 97366. The DNA segment frompML103 contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kDzein gene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize10 kD zein gene in the vector pGem9Zf(+) (Promega). Vector and insertDNA can be ligated at 15° C. overnight, essentially as described(Maniatis). The ligated DNA may then be used to transform E. coliXL1-Blue (Epicurian Coli XL-1 Blue™; Stratagene). Bacterialtransformants can be screened by restriction enzyme digestion of plasmidDNA and limited nucleotide sequence analysis using the dideoxy chaintermination method (Sequenase™ DNA Sequencing Kit; U.S. Biochemical).The resulting plasmid construct would comprise a recombinant DNAconstruct encoding, in the 5′ to 3′ direction, the maize 27 kD zeinpromoter, a cDNA fragment encoding the instant polypeptides, and the 10kD zein 3′ region.

The recombinant DNA construct described above can then be introducedinto corn cells by the following procedure. Immature corn embryos can bedissected from developing caryopses derived from crosses of the inbredcorn lines H99 and LH132. The embryos are isolated 10 to 11 days afterpollination when they are 1.0 to 1.5 mm long. The embryos are thenplaced with the axis-side facing down and in contact withagarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking18:659-668). The embryos are kept in the dark at 27° C. Friableembryogenic callus consisting of undifferentiated masses of cells withsomatic proembryoids and embryoids borne on suspensor structuresproliferates from the scutellum of these immature embryos. Theembryogenic callus isolated from the primary explant can be cultured onN6 medium and sub-cultured on this medium every 2 to 3 weeks.

The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

The particle bombardment method (Klein et al. (1987) Nature 327:70-73)may be used to transfer genes to the callus culture cells. According tothis method, gold particles (1 μm in diameter) are coated with DNA usingthe following technique. Ten μg of plasmid DNAs are added to 50 μL of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μL ofa 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After 10 minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains gluphosinate (2 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additional2 weeks the tissue can be transferred to fresh N6 medium containinggluphosinate. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing theglufosinate-supplemented medium. These calli may continue to grow whensub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 6 Expression of Recombinant DNA Constructs in Dicot Cells

A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the 13 subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptides in transformed soybean. The phaseolincassette includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Nco I (whichincludes the ATG translation initiation codon), Sma I, Kpn I and Xba I.The entire cassette is flanked by Hind III sites.

The cDNA fragment of this gene may be generated by polymerase chainreaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

Soybean embryos may then be transformed with the expression vectorcomprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can maintained in 35 mL liquidmedia on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a16:8 hour day/night schedule. Cultures are subcultured every two weeksby inoculating approximately 35 mg of tissue into 35 mL of liquidmedium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein et al. (1987) Nature (London)327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene which can be used to facilitate soybeantransformation is a recombinant DNA construct composed of the 35Spromoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812), the hygromycin phosphotransferase gene from plasmid pJR225(from E. coli; Gritz et al. (1983) Gene 25:179-188) and the 3′ region ofthe nopaline synthase gene from the T-DNA of the Ti plasmid ofAgrobacterium tumefaciens. The seed expression cassette comprising thephaseolin 5′ region, the fragment encoding the instant polypeptides andthe phaseolin 3′ region can be isolated as a restriction fragment. Thisfragment can then be inserted into a unique restriction site of thevector carrying the marker gene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi and the chamber is evacuated to a vacuum of 28 inchesmercury. The tissue is placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue can be divided in half and placed back into liquid and culturedas described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post bombardment with freshmedia containing 50 mg/mL hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 7 Expression of Recombinant DNA Construct in Microbial Cells

The cDNAs encoding the instant polypeptides can be inserted into the T7E. coli expression vector pBT430. This vector is a derivative of pET-3a(Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

Plasmid DNA containing a cDNA may be appropriately digested to release anucleic acid fragment encoding the protein. This fragment may then bepurified on a 1% NuSieve GTG™ low melting agarose gel (FMC). Buffer andagarose contain 10 μg/ml ethidium bromide for visualization of the DNAfragment. The fragment can then be purified from the agarose gel bydigestion with GELase™ (Epicentre Technologies) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs, Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptides are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

For high level expression, a plasmid clone with the cDNA insert in thecorrect orientation relative to the T7 promoter can be transformed intoE. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol.189:113-130). Cultures are grown in LB medium containing ampicillin (100mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG(isopropylthio-β-galactoside, the inducer) can be added to a finalconcentration of 0.4 mM and incubation can be continued for 3 h at 25°.Cells are then harvested by centrifugation and re-suspended in 50 μL of50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride. A small amount of 1 mm glass beads can be addedand the mixture sonicated 3 times for about 5 seconds each time with amicroprobe sonicator. The mixture is centrifuged and the proteinconcentration of the supernatant determined. One μg of protein from thesoluble fraction of the culture can be separated by SDS-polyacrylamidegel electrophoresis. Gels can be observed for protein bands migrating atthe expected molecular weight.

Example 8 Evaluating Compounds for Their Ability to Inhibit the Activityof Ammonium Transporters

The polypeptides described herein may be produced using any number ofmethods known to those skilled in the art. Such methods include, but arenot limited to, expression in bacteria as described in Example 7, orexpression in eukaryotic cell culture, in planta, and using viralexpression systems in suitably infected organisms or cell lines. Theinstant polypeptides may be expressed either as mature forms of theproteins as observed in vivo or as fusion proteins by covalentattachment to a variety of enzymes, proteins or affinity tags. Commonfusion protein partners include glutathione S-transferase (“GST”),thioredoxin (“Trx”), maltose binding protein, and C- and/or N-terminalhexahistidine polypeptide (“(His)₆”). The fusion proteins may beengineered with a protease recognition site at the fusion point so thatfusion partners can be separated by protease digestion to yield intactmature enzyme. Examples of such proteases include thrombin, enterokinaseand factor Xa. However, any protease can be used which specificallycleaves the peptide connecting the fusion protein and the enzyme.

Purification of the instant polypeptides, if desired, may utilize anynumber of separation technologies familiar to those skilled in the artof protein purification. Examples of such methods include, but are notlimited to, homogenization, filtration, centrifugation, heatdenaturation, ammonium sulfate precipitation, desalting, pHprecipitation, ion exchange chromatography, hydrophobic interactionchromatography and affinity chromatography, wherein the affinity ligandrepresents a substrate, substrate analog or inhibitor. When the instantpolypeptides are expressed as fusion proteins, the purification protocolmay include the use of an affinity resin which is specific for thefusion protein tag attached to the expressed enzyme or an affinity resincontaining ligands which are specific for the enzyme. For example, theinstant polypeptides may be expressed as a fusion protein coupled to theC-terminus of thioredoxin. In addition, a (His)₆ peptide may beengineered into the N-terminus of the fused thioredoxin moiety to affordadditional opportunities for affinity purification. Other suitableaffinity resins could be synthesized by linking the appropriate ligandsto any suitable resin such as Sepharose-4B. In an alternate embodiment,a thioredoxin fusion protein may be eluted using dithiothreitol;however, elution may be accomplished using other reagents which interactto displace the thioredoxin from the resin. These reagents includeβ-mercaptoethanol or other reduced thiol. The eluted fusion protein maybe subjected to further purification by traditional means as statedabove, if desired. Proteolytic cleavage of the thioredoxin fusionprotein and the enzyme may be accomplished after the fusion protein ispurified or while the protein is still bound to the ThioBond™ affinityresin or other resin.

Crude, partially purified or purified enzyme, either alone or as afusion protein, may be utilized in assays for the evaluation ofcompounds for their ability to inhibit enzymatic activation of theinstant polypeptides disclosed herein. Assays may be conducted underwell known experimental conditions which permit optimal enzymaticactivity.

Transformation of Saccharomyces cerevisiae ammonium transport mutant26972c, which lacks high affinity ammonium transporters, with theinstant cDNAs encoding ammonium transporters will evaluation ofcompounds for their ability to inhibit enzymatic activation of theinstant polypeptides disclosed herein. Assays may be conducted underwell known experimental conditions to test for viability of thetransformed yeast stain as presented by Marini et al. (1997). Mol. Cell.Biol. 17:4282-4293.

1. An isolated polynucleotide comprising: (a) a nucleotide sequenceencoding a polypeptide having ammonium transporter activity, wherein theamino acid sequence of the polypeptide and the amino acid sequence ofSEQ ID NO:16 have at least 95% sequence identity based on the Clustalalignment method, or (b) the complement of the nucleotide sequence,wherein the complement and the nucleotide sequence contain the samenumber of nucleotides and are 100% complementary.
 2. The polynucleotideof claim 1 wherein the polypeptide comprises the amino acid sequence ofSEQ ID NO:16.
 3. The polynucleotide of claim 1 wherein the nucleotidesequence comprises the nucleotide sequence of SEQ ID NO:15.
 4. A vectorcomprising the polynucleotide of claim
 1. 5. A recombinant DNA constructcomprising the polynucleotide of claim 1 operably linked to a regulatorysequence.
 6. A method for transforming a cell comprising transforming acell with the polynucleotide of claim
 1. 7. A cell comprising therecombinant DNA construct of claim
 5. 8. A method for producing a plantcomprising transforming a plant cell with the polynucleotide of claim 1and regenerating a plant from the transformed plant cell.
 9. A plantcomprising the recombinant DNA construct of claim
 5. 10. A seedcomprising the recombinant DNA construct of claim 5.